Mutations generate the genetic diversity that is essential for life. The ability to create genetic variation also comes at a price, with the potentil for detrimental consequences. The fidelity of genomic information must be meticulously preserved during transcription and replication, to avoid deleterious outcomes of unintended changes to DNA sequences. Although avoidance of mutation and DNA damage is key to maintaining genomic integrity, the human genome encodes multiple enzymes that are employed intentionally to induce mutations and double-strand breaks (DSBs) in genomic DNA. The human immune system employs such DNA editing enzymes to combat the large array of pathogens to which we are exposed. For example, the activation-induced deaminase (AID) is a central mediator of the adaptive immune response, and drives antibody diversification. The related cytosine deaminases of the APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide 3) subfamily mutate virus genomes as part of the intrinsic cellular antiviral defense. The beneficial effects of these powerful mutators of viral DNA must be balanced with their potential to act on the host genome. We propose that human APOBEC3 (hA3) enzymes threaten host genome integrity, and could be the source of DSBs and clustered somatic mutations in human cancers. The ability of hA3 proteins to edit cellular DNA implicates them in driving genomic instability and provides a compelling reason to investigate how their mutator activity is regulated. Our long-term goal is to understand how hA3 enzymes are regulated to achieve efficient restriction of virus genomes, while limiting the potential for oncogenic lesions n cellular DNA. We focus on two hA3 members (hA3A and hA3B) that can both be localized in the nucleus and have a mutational signature that matches that seen with cytosine transitions in cancer genomes. Supported by strong preliminary data, this proposal will determine how the cellular genome is susceptible to damaging effects of hA3A/hA3B activity. Our Specific Aims will be: (i) To determine how hA3 enzymes deaminate the genome and cause DSBs, (ii) To determine the extent to which hA3 proteins generate clustered mutations at DSBs in human cells, and (iii) To determine what regulates hA3A/hA3B activity. The proposed research into hA3 enzymes as potential sources for somatic mutations in human cancers, represents an innovative departure from previous studies focused on antiviral activities of these enzymes. The results of this proposal will establish mechanisms for hA3 action on the cellular genome and processes that regulate their activity. RELEVANCE TO HUMAN HEALTH: Our studies form the foundation in a continuum of research that is expected to reveal how misregulation of hA3 mutator enzymes contributes to human cancers. Such knowledge has the potential to lead to novel strategies for therapeutic interventions to limit somatic mutations that drive cancer progression.